J Pharmacol Sci 97, 489 – 493 (2005) Journal of Pharmacological Sciences

©2005 The Japanese Pharmacological Society

Full Paper

A Theoretical Method for Normalizing Total Serum Valproic Acid

Concentration in Hypoalbuminemic Patients
Jesús Hermida1 and J. Carlos Tutor1,*
1
Central Laboratory, University Hospital Clinic, 15706 Santiago de Compostela, Spain

Received December 21, 2004; Accepted January 27, 2005

Abstract. In patients with hypoalbuminemia, the total serum concentration of valproic acid
may offer poor clinical information; however, very few clinical laboratories routinely analyze the
free concentration of the drug. The aim of this study was to design a procedure to normalize the
total concentration of valproic acid according to the level of serum albumin and using previously
published free fraction values. In 121 adult patients, with albumin levels of 18 – 41 g / L, the
total concentration of valproic acid was normalized using the derived equation: CN = a HCH / 6.5,
where a H is the free fraction of the drug corresponding to the patient’s particular albuminemia
and CH is the total concentration of valproic acid. The value of 6.5 corresponds to the free
fraction of the drug for a serum albumin of 42 g / L (percentile 50 of the reference range). For
total concentrations lower than 75 mg / L, the predicted normalized valproic acid concentrations
were reasonably concordant with the observed normalized concentrations calculated using the
data from a protein-binding study. In a significant number of cases, subtherapeutic concentrations
of the drug became therapeutic and even supratherapeutic when corrected according to the
albumin levels. Furthermore, cases with therapeutic drug concentrations frequently became
supratherapeutic when normalized. The limitations and clinical aplications of the proposed
formula for normalizing the total concentration of valproic acid are presented. It is concluded
that it may be useful for the posological management of hypoalbuminemic patients when the
free concentration of the drug is not available, and decisions have to be made based on the total
serum concentration.

Keywords: valproic acid, hypoalbuminemia, dose adjustment, ultrafiltration

Introduction (a <20%), it may be preferable to determine their free

concentration; however, in clinical laboratories, the
In blood, most drugs are bound to serum proteins, standard procedure is to monitor the total drug concen-
principally albumin, a1-acid glycoprotein and lipo- tration (1 – 4), due to technical difficulties and a lack of
proteins, to various degrees. The fraction bound to established reference ranges for free drug concentrations
proteins is considered to be pharmacologically inactive, (2, 4). The two antiepileptic drugs in which it may be
as only the unbound (free) fraction is available for clinically useful to determine the free drug concentration
diffusion out of the vascular system to sites for are phenytoin and valproic acid (5, 6). Free phenytoin is
pharmacological action (1 – 4). The free fraction (a ) the most frequently requested free drug concentration by
represents the relationship between free and total drug clinicians (3, 5), possibly because they are more familiar
concentration: with it (5).
Valproic acid is extensively bound to albumin
a = Free drug concentration / Total drug concentra-
(a <10%), and the drug-protein binding depends on
tion (free + bound)
both the serum albumin concentration and the drug
In the case of drugs with high protein binding concentration (2 – 7). As a result, the free fraction of
this drug is subject to more variation than other highly
*Corresponding author. FAX: +34 981 950403
E-mail: [email protected], [email protected]
protein-bound antiepileptic drugs (2). Studies published

489
490 J Hermida and JC Tutor

by Gidal et al. (8) and Haraldson et al. (9) reported committee of the University of Santiago de Compostela
several cases demonstrating the clinical importance of Hospital Clinic, and all participants provided their con-
monitoring free valproic acid, as well as the minimal sent to participate.
utility of total drug concentration in patients with The determination of free and total valproic acid
hypoalbuminemia. As early as 1980, Levy stressed that was carried out by fluorescence polarization immuno-
serious consideration should be given to the monitoring assay in a Cobas Integra 400 analyzer using reagents
of free rather than total valproic acid concentration, commercialized by Roche Diagnostics (Basel, Switzer-
which can be misleading (7); however, a recent survey land). The sensitivity of the free valproic acid assay is
by the College of American Pathologists revealed that 1.3 mg / L and its determination was made in duplicate.
only 2% of the laboratories which routinely performed Protein-binding of valproic acid was evaluated by ultra-
total valproic acid determination offered free valproic filtration at 25°C using the Centrifree Micropartition
acid assays (2). For some anticonvulsant drugs, under System (Millipore Corporation, Bedford, MA, USA)
well-controlled and standardized conditions, the concen- for the unbound drug separation (16). Serum albumin
tration in saliva bears a constant relationship to free was determined using bromocresol purple (17) in a
serum concentration, but this is not the case for valproic Dade Dimension analyzer (Dade Behring, Liederbach,
acid (10). Germany).
The ability of equations using population mean bind- In hypoalbuminemic patients, the free fraction of
ing parameters (association constant and total concentra- valproic acid (a H) may be given by the equation:
tion of binding sites) to predict unbound valproic acid a H = CF / CH, where CF is the free drug concentration
concentration was studied by Kodama et al. in pediatric and CH is the total concentration of the drug. In
and adult patients, whose albumin concentrations were conditions of normoalbuminemia, for the same free
assumed to be normal (11 – 13). However, because concentration of the drug (CF), the free fraction (a N)
albumin levels directly influence the binding of the drug may be a N = CF / CN, where CN is the total normalized
to plasma proteins, these methods may not accurately concentration of valproic acid. Combining both equa-
predict unbound valproic acid in patients with hypo- tions: a NCN = a HCH and CN = a HCH / a N. This formula
albuminemia. The relationship obtained by Parent et al. may be used to determine the total drug concentration
(14) between the free fraction of valproic acid (y) and that would be expected if the patients’ albumin concen-
serum albumin concentration (x): y = Ae-Bx (A = 130.69, trations were normal.
B = 4.96 ´ 10-3, r = -0.82) would make it possible to The normalized concentration of valproic acid for a
estimate the free concentration of the drug. In our paper, serum albumin of 42 g / L is given by the formula:
a derived formula is proposed for normalizing the total CN = a HCH / 6.5, where 6.5 is the free fraction of the drug
concentration of valproic acid in hypoalbuminemic for this albumin concentration (Table 1), in agreement
patients. with the results presented by Parent et al. (14). The total
experimental concentrations of valproic acid for all of
Materials and Methods the patients studied were normalized using this formula.
Statistical analysis of the data was carried out using the
Total serum concentrations of valproic acid were Microsoft Excel (v.5.0) package, and the Kolmogorov-
determined for 121 adult patients who were receiving Smirnov test was used to check for normality. As the
anticonvulsant treatment with this drug, all of who had a data had not a Gaussian distribution, the Spearman’s
concentration of serum albumin lower than 42 g / L correlation coefficient and Passing-Bablock regression
(mean 35.5 ± 4.6 g / L, range 18 – 41 g / L), correspond- method were used.
ing to percentile 50 of the reference range for individuals
between the ages of 25 – 55 years (15). In 53 patients, Results
with a mean serum albumin concentration of 36.9 ±
5.0 g / L (range 24 – 41 g / L) and normal levels of In the 53 patients in which the protein-binding of
bilirubin, creatinine, and urea, who were treated in valproic acid was evaluated, for total drug concentra-
monotherapy (n = 37) or in polytherapy with phenytoin, tions lower than 75 mg / L (n = 42), the means of the
carbamazepine, lamotrigine, or mysoline (n = 16), predicted (using Table 1) and observed free fractions
additionally to the total, the free concentrations of were analogous (10.6 ± 4.8% vs 10.1 ± 6.3%). However,
valproic acid were determined by ultrafiltration. Blood for total concentrations greater than 75 mg / L (n = 11),
samples were taken immediately before the morning the predicted free fractions (7.7 ± 1.3%) were signifi-
dose (trough level) once the state of equilibrium had cantly lower (P<0.001) than the observed free fractions
been reached. The study was approved by the ethical (13.8 ± 3.2%). The scatter diagram of the unbound
 Valproic Acid Levels in Hypoalbuminemia 491

Table 1. Relationship between free fraction of valproic acid (a)

and serum albumin concentration (Ref. 14)
Albumin (g/L) a (%)

42 6.5
41 6.8
40 7.3
39 7.9
38 8.5
37 9.1
36 9.8
35 10.5
34 11.3
33 12.1
32 13.0
31 14.0
30 15.0
29 16.2
28 17.4
27 18.7
Fig. 1. Scatter diagram of unbound valproic acid prediction error
26 20.1 against total valproic acid concentration.
25 21.6
24 23.2
23 24.9
22 26.8
21 28.9
20 31.0
19 33.3
18 35.8

valproic acid prediction error (difference between

predicted and observed unbound concentrations) against
total valproic acid concentrations, reveal that this error
was £2.5 mg/ L for total drug concentrations lower than
aproximately 75 mg / L (see Fig. 1); however, for greater
total concentrations, the unbound prediction error in-
creases significantly. Figure 2 indicates the relationship
between predicted and observed normalized valproic
acid concentrations, using the proposed formula and
the predicted and observed free fractions, respectively,
for total drug concentrations lower and greater than Fig. 2. Relationship between predicted and observed normalized
75 mg / L. valproic acid concentrations for total valproic acid concentrations
Figure 3 shows the relationship between predicted lower (open circles) and higher (closed circles) than 75 mg/ L.
normalized and total valproic acid concentrations in the
group of 121 patients. In these patients, 48 cases had
subtherapeutic total valproic acid concentrations, with Discussion
47.9% of these cases reaching therapeutic levels and
10.4% supratherapeutic levels after being normalized for The relationship indicated by Parent et al. between the
albumin concentrations. Similarly, in the 65 patients that free fraction of the valproic acid and serum albumin
have therapeutic total concentrations of the drug, 47.7% concentration, in the absence of renal failure, jaundice,
reached supratherapeutic levels after normalization. or a high total drug concentration, would make it
492 J Hermida and JC Tutor

In cases of neurotoxicity previously described in hypo-

albuminemic patients, with high free concentrations of
valproic acid (8, 9), normalization of the total concentra-
tion of the drug would have offered clinically useful
information (CN >100 mg / L), suggesting a decrease of
the doses.
The valproic acid free fractions used for the calcula-
tion of the normalized concentrations (Table 1), are
reasonably concordant with the in vitro values recently
obtained by Bailey and Briggs for a total drug concentra-
tion of 75.0 mg / L (18), and the only criteria used to
select the 121 patients was that they presented a serum
albumin level of less than 42 g / L. Changes in a 1-acid
glycoprotein concentrations had little effect (18);
however, other factors apart from the albumin concen-
tration may affect the free fraction of valproic acid.
The protein binding is also drug-concentration-depen-
dent, with a free fraction increasing from approximately
5% at a total valproic acid concentration of 10 – 60
mg / L to 10% at 50 – 100 mg / L and 20 – 30% at 145 –
Fig. 3. Scatter diagram of predicted normalized valproic acid
concentrations against total valproic acid concentrations. The dashed
160 mg / L (3, 4, 7), exhibiting a saturable protein
lines correspond to the limits of therapeutic range. binding at the upper end of the therapeutic range (2, 5).
As a result, there is an unpredictable free fraction at high
total drug concentrations. However, it is important to
possible to estimate the free drug concentration with an take into account that it is the normalization of total
error of £2.5 mg / L (14). Our results appear to confirm subtherapeutic and therapeutic concentrations of valproic
this aspect for total valproic acid concentrations acid that would be of real clinical interest, in which the
£75 mg / L (see Fig. 1). Consequently, the predicted and imprecision of the free drug concentration determination
observed normalized concentrations were reasonably is greater.
concordant for total valproic acid concentrations lower The most widely-used antiepileptic drugs (phenytoin,
than 75 mg / L (Fig. 2), although a greater unbound phenobarbital, and carbamazepine) have no effect on
prediction error would be produced for total concentra- valproic acid binding to albumin (1), and salicylate is the
tions around this cut-off value in very severe cases of only acidic drug know to effectively compete with
hypoalbuminemia. For a greater total concentration valproic acid for albumin binding (4, 5). However,
(80 – 120 mg / L), the underestimation of the predicted endogenous substances such as free fatty acids, uremic
normalized concentration is evident (Fig. 2); however, compounds and bilirubin would displace the bound
in this case, given the discrete dispersion of the values, drug from its albumin binding sites (1). Therefore, in
the predicted normalized concentration may be corrected situations of jaundice or uremia, which may produce
using the corresponding linear regression equation. The an increase of the free drug fraction, the application of
higher dispersion found between the values of predicted the derived formula could lead to an underestimation of
and observed normalized concentrations for total the normalized concentration of valproic acid.
valproic acid concentrations lower than 75 mg / L, may The clinicians are much more familiar with the use of
be due to a higher imprecision in the experimental the total than free serum drug concentrations. Evidently,
determination of lower free drug concentrations. in the best of cases the predicted normalized total
As may be seen from the results indicated above valproic acid concentrations offer approximate results.
(Fig. 3), normalization of the total concentration of However, despite its obvious limitations, the proposed
valproic acid in patients with hypoalbuminemia may formula may be of clinical use for dealing with hypo-
have a significant influence on adjusting the dose of the albuminemic patients in centres where the free valproic
drug. In patients with subtherapeutic total concentra- acid concentration is not available, and posological
tions, after correcting these according to the albumin decisions have to be made based on the total concentra-
concentration, they frequently became therapeutic and tion of the drug. An evaluation of its clinical perfor-
even supratherapeutic. Similarly, therapeutic levels mance is currently underway.
frequently became supratherapeutic after normalization.
 Valproic Acid Levels in Hypoalbuminemia 493

Acknowledgments using saliva samples. Clin Pharmacokinet. 1999;36:453–470.

11 Kodama Y, Kuranari M, Kodama H, Koike Y, Yasunaga F, Fujii
I, et al. Evaluation of binding equation method for prediction of
The authors would like to thank Victoria Lourido,
unbound serum valproic acid concentration in pediatric patients
MB, by her collaboration. with epilepsy. Int J Clin Pharmacol Ther. 1995;33:114–118.
12 Kodama Y, Kuranari M, Kodama H, Ashikari Y, Fujii I,
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